The commonly used means to differentiate between normal and non-normal states of a human subject using his or her biological materials are mainly those which have been used in the field of diagnostics. Most frequently used are those methods which target biomarkers in blood. It has been practiced in this field to comparatively measure the amount of a specific protein or a peptide that is less than 10,000 in molecular weight or, in the case of enzyme protein, enzyme activities in samples from normal (healthy) subjects and those from diseased individuals to help diagnosis. Here, prior to testing real samples, measurements are done on a fixed number each of samples from healthy controls and patients with certain disease with respect to the amount (s) or activity (activities) of single or multiple specific proteins or peptides and the ranges of abnormal and normal values are respectively determined. The sample to be evaluated is then analyzed by the same method and the resultant value is judged with respect to whether it is in normal or in abnormal range.
In the actual measurements, the amount(s) of specified protein(s) or peptide(s) in test samples, as such or after dilution, are determined by the use of enzyme-linked immunosorbent assay (ELISA) which uses a primary, or secondary, antibody labeled with an enzyme reacting with a substrate that yields a color upon reaction, chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA) which uses a primary, or secondary, antibody labeled with a radioisotope, and, if the protein is an enzyme, the measurement of the activity of the enzyme by adding its substrate and determining the intensity of produced color, etc. These antibody-based methods are called as enzyme-, fluorescence- or radioisotope-labeled methods, respectively. In addition, there is a method where an enzyme reaction product derived from the corresponding substrate is determined by high performance liquid chromatography (HPLC). In further addition, there is a method where HPLC is combined with mass spectrometer, called LC-MS/MS, and there is a method called selected reaction monitoring (SRM)/multiple reaction monitoring (MRM) that utilizes LC-MS/MS. In another method to determine the concentration in a sample, it is appropriately pretreated, and separation of proteins or peptides is attained by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and target protein or peptide is determined by silver staining, Coomassie blue staining or immunological staining (Western blotting) that uses an antibody to target protein or peptide. In still further addition, there is a method which utilizes mass spectrometry to determine the amount of target protein or peptide in samples fractionated by column chromatography. Instead of column chromatography, protein chips and magnetic beads may also be utilized for purpose of pretreatment.
Furthermore, these inventors have developed an immunoMS method, where target protein or peptide is captured by beads (including magnetic ones) with linked antibody to the protein or peptide, eluted from the beads, and determined by mass spectrometry. Further, intact proteins have been reported to be analyzed by mass spectrometry using above-mentioned methods after digestion with trypsin etc. (PTL 1). Here, intact target proteins are selected either by fractionation or by adsorption to an adsorbent specific to them and then determined by mass spectrometry.
Number of patients suffered from cognitive impairment like Alzheimer disease is increasing rapidly along with increasing of old-age population in Japan. It is estimated that number of patients is 1.3 million in 1995 and it will be 1.9 million in 2005 and will reach to about 3.0 million in 2020. It is reported that 60-90% of cognitive impairment is Alzheimer disease. As manifestation of Alzheimer disease is not only loss of memory but several disturbance in daily life, increase of patients of this disease is becoming an important social issue to be solved. In Japan, Donepezil-hydrochloride, anti-acetylcholine esterase inhibitor has been available for medical treatment for Alzheimer disease since 1999, and it let progress of cognitive impairment in these patients be ‘slow-down’ efficiently, if the patient is diagnosed at early stage. Thus, in medication of Alzheimer disease, most important issue is ‘early diagnosis’ to treat the patients effectively by drug available at present and new coming drug.
Followings are major criteria for diagnosis of Alzheimer disease described in DSM-IV, which is published by American Psychiatric Association (Non-patent reference 1).                A. The development of multiple cognitive deficits manifested by both                    (1) memory impairment (impaired ability to learn new information or to recall previously learned information)            (2) one (or more) of the following cognitive disturbances:                            a) aphasia (language disturbance)                b) apraxia (impaired ability to carry out motor activities despite intact motor function)                c) agnosia (failure to recognize or identify objects despite intact sensory function)                d) disturbance in executive functioning (i.e., planning, organizing, sequencing, abstracting)                                                B. The cognitive deficits in Criteria A1 and A2 each cause significant impairment in social        or occupational functioning and represent a significant decline from a previous level of functioning.        
There are several types of neurological disorders related to Alzheimer disease (AD). As cognitive dysfunction appears gradually in dementia including AD, there is a disease status of pre-stage of dementia. This stage is called as mild cognitive impairment (MCI). In United States, 10% MCI develops to AD within 1 year, and 50% of MCI develops to AD within 4 years. MCI is defined as a condition characterized by newly acquired cognitive decline to an extent that is beyond that expected for age or educational background, yet not causing significant functional impairment, and not showing disturbance in daily life. Frontotemporal dementia (frontotemporal lobar degeneration) (FTD) shows loss of personal awareness, loss of social awareness, hyperorality, and stereotyped, perseverative behavior. These clinical characteristics are different from AD. FTD includes Pick's disease, which is characterized by microscopically Pick bodies usually found in limbic, paralimbic, and ventral temporal lobe cortex. Dementia with Lewy bodies (DLB) is characterized by progressive disease and psychiatric symptoms include anxiety, depression, hallucinations (usually visual) and delusions (false beliefs). DLB is thought to be the second most common subtype and 10-30% of dementia is DLB. The symptoms of DLB are caused by the build-up of Lewy bodies histologically. FTD and DLB belong to demented neurological disease as they also lose of memory, their ability to solve problems and maintain emotional control (NPL 1). In description in present patent, cognitive impairment includes AD, MCI and the demented neurological disease.
The screening tests for dementia widely used are the Hasegawa Dementia Scale-revised (HDS-R) and Mini-Mental State Examination (MMSE). In these screening tests, inspector asks several questions and evaluates level of cognitive impairment of each subject by scores. HDS-R is revised version of HDS published in 1991. In HDS-R, test consists 9 questions to analyses orientation, remembrance, calculation, retain and recall ability, and common sense. Full score is 30 and a person whose score is less than 23 is suspected as dementia. MMSE has been developed in United States to screen and diagnose dementia, and analyses global cognitive function, with items assessing orientation, word recall, attention and calculation, language abilities, and visuospatial (drawing) ability. This test consists of 11 questions, and full score is 30 and a person who has score less than 23 is suspected as dementia. The results of HDS-R and MMSE coincide with each other. Both are used for screening, not for diagnosis and not for staging of disease progression (NPL 1).
Neuroimaging test for dementia are Computed tomography (CT) and Magnetic resonance imaging (MRI) which evaluate morphological changes like brain atrophy and ventricular dilation and single-photon emission computed tomography (SPECT) which analyses regional cerebral blood flow and PET which shows brain metabolism by measurement of consumption of oxygen and sugar. SPECT and PET, nuclear imaging technologies, can identify neuronal dysfunction at preclinical stage (NPL 1). However, these neuroimaging can not be widely used in hospitals because they need special facilities to perform nuclear imaging, and neuroimaging may not be objective test as imaging diagnosis is completely depend on the skill of physician who analyses the mages.
Thus, methods for screening and diagnosis of dementia including AD that are available at present is dependent on tests lacking objectivity and is dependent on expensive instruments, and so it is very difficult to use these tests for screening of early stage-cognitive impairment. If we get blood (serum/plasma) biomarker for cognitive impairment, which enables us objective test using specimens we can easily obtain, we can identify cognitive impairment at early stage (preclinical stage) by blood test using such biomarker. Present patent provides novel biomarkers and a novel and potent diagnostic method for cognitive impairment by using such biomarkers and biomarkers described here. In addition, present patent provides diagnostic method and novel biomarkers for non-demented neurological disease like depression, schizophrenia, etc.